PSGL-1 is a selectin ligand on leukocytes that plays a critical role in inflammation. The current application represents a continuation of studies that address the biology of P-selectin and PSGL-1 and seeks to identify and isolate the second physiologic counterreceptor for E-selectin in addition to PSGL-1. With the PSGL-1 null mouse in hand prepared during the past grant period, we have used this mouse to prove that (1) PSGL-1 is the dominant P-selectin ligand in vivo; (2) PSGL-1 is a physiologic ligand for E-selectin; and (3) there is a second, physiologically relevant E-selectin ligand. Using systems to identify the E-selectin ligand under flow conditions, we are now positioned to determine whether ESL-1 is the second E-selectin ligand or to identify a novel E-selectin ligand. Antibodies to ESL-1 will be used in vivo to determine whether, in the absence of PSGL-1, they inhibit E-selectin-dependent leukocyte rolling. Furthermore, WEHI cells lacking PSGL-1 and ESL-1 will be developed using RNAi. The interaction of E-selectin with a 2D protein blot of these WEHI cells under rolling conditions will identify bound cells to a protein spot using difference imaging gel electrophoresis. The protein spot of interest will be identified by mass spectroscopy. New initiatives in this proposal employ the novel intravital microscopy system that we have developed. This system allows collection of real time confocal or widefield images of the microcirculation of a living mouse from three fluorescence channels and one brightfield channel simultaneously. Using intravital imaging, we show that microparticle rolling on the venule vascular endothelium is one of the first steps during the inflammatory response. This new observation will seed an initiative to understand the function of PSGL-1-bearing microparticles during inflammation. This potentially represents a system for the delivery of leukocyte components to inflammatory sites and/or fusion of microparticle membranes to cell membranes of cells associated with the vessel wall. Furthermore, we will use the intravital microscopy system to explore leukocyte rolling, with specific focus on the transfer of membrane and membrane proteins from one cell surface to another in vivo. Finally, we propose to use the PSGL-1 null mouse to establish whether the absence of PSGL-1 will block or mitigate graft versus host disease following bone marrow transplantation in a mouse model.